IgG is a major protein of serum, and plays an important role in recognizing and eliminating foreign matter in the immune system. Making use of this characteristic, IgG is widely studied for applications to therapeutic drugs and diagnostic reagents for various diseases, and test reagents. Such applications include antibody therapies for cancer; therapies based on antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), molecular-targeted drugs that specifically block and starve receptors and the like expressed in cancer cells by means of antibodies, or missile therapy based on cancer cell specific antibody coupled with an anticancer agent, and the like are under development. Amid this situation, an anti-HER2 receptor humanized monoclonal antibody was developed and launched as a therapeutic agent for malignant tumors such as breast cancer (trade name Herceptin). IgG is also used as an essential tool for a range of biochemical experiments, including immunoassay, cell or protein functional analysis, and gene expression screening, on the basis of its property of specific binding to antigens.
IgG has a Y-shaped structure wherein two H chains and two L chains are bound via disulfide bonds (S—S bonds). When decomposed with the proteinase papain, IgG can be divided into an Fc fragment, which consists of a constant region, and a Fab fragment, which comprises an antigen-binding site. IgG involves subclasses; in the case of human IgG, there are four subclasses IgG1, IgG2, IgG3, and IgG4.
Antibodies are purified from serum or hybridoma cell culture supernatant liquid using a column for antibody purification. Generally, for the first-stage purification, Protein A is used as the ligand. Protein A is a protein with a molecular weight of 42 kDa, produced by Staphylococcus aureus, and binds strongly to the Fc region of IgG. Protein A is expensive, and there are some cases in which highly pure antibody cannot be obtained because of animal species or subclass, or in which antibodies undergo denaturation under purification conditions with the use of Protein A; there is a demand for a novel separating agent with higher performance than that of Protein A.
Antibodies labeled with fluorescent substances or enzymes are used in a range of experiments, including immunohistochemical experiments, histological staining, ELISA, Western blotting, flowcytometry and the like. For example, in histological staining, by using an antibody having a fluorescent substance such as FITC bound thereto, the tissue localization of desired protein can be examined. In assays such as ELISA and Western blotting, more sensitive assays can be performed by first reacting a primary antibody to the substance to be detected, then reacting a labeled secondary antibody that binds to the primary antibody. For example, in the ECL system from GE Healthcare, an antibody having horseradish peroxidase bound thereto is used as the secondary antibody, and luminol is oxidized and allowed to produce light by the catalytic action of the horseradish peroxidase, whereby the desired substance is detected. However, it takes much labor and time to bind a labeling substance to an antibody by chemical modification, and the antibody sometimes undergoes denaturation; there is a need for the development of a novel technology for antibody labeling. For labeled secondary antibodies, there is a demand for less expensive ones with higher sensitivity.
Development of antibody chips as diagnostic chips for various diseases is ongoing. One problem to be solved is to develop a method for immobilizing an antibody to a substrate, wherein the antigen binding sites of the antibody are arranged at high density in a highly active state on the surface of the substrate. In methods of immobilization utilizing non-specific adsorption and methods of immobilization utilizing amino groups, antibody molecules become arranged randomly so that no sufficient sensitivity can be obtained.
Research and development for antibodies have been rapidly promoted for use as molecular-targeted therapeutic drugs for diseases such as cancer and rheumatism; about 20 kinds of antibody drugs have been brought into practical applications to date, and clinical studies of about 300 kinds of antibody drug candidates are underway worldwide. Initially in the development, mouse monoclonal antibodies were used as antibody drugs; however, because mouse antibodies were recognized as foreign matter by the human immune system and production of human anti-mouse antibodies was induced, no sufficient therapeutic effect could be achieved. Hence, using gene recombination technology, chimeric antibodies wherein the constant regions of mouse antibodies were replaced with the constant regions of human antibodies and humanized antibodies wherein all portions, but the complementarity determinant regions, of mouse antibodies were replaced with human antibodies were developed. A method for preparing a human monoclonal antibody using a human antibody-producing mouse (KM mouse) has also been developed.
One of monoclonal antibody drugs used for antibody therapy is prepared by binding an anticancer agent or toxin to an antibody that specifically recognizes cancer cells, and this is internalized in target cells to kill the target cells. The anticancer agent or toxin needs to be detached from the antibody after internalization. For this reason, a manipulation is made to allow the anticancer agent or toxin to be detached from the antibody after internalization by, for example, adding a protease recognition site to the linker that binds the antibody and the anticancer agent or toxin. For example, gemtuzumab ozogamicin (Mylotarg), which has been developed as a therapeutic drug for acute myelocytic leukemia, comprises a humanized anti-CD33 monoclonal antibody and a calicheamicin derivative bound thereto; when Mylotarg binds to CD33 and becomes internalized in cells, the calicheamicin derivative is liberated to kill the cells. Hence, it is important to design a linker that binds an antibody and an anticancer agent or toxin; to achieve higher pharmacological efficacy, development of novel linkers is ongoing.
In recent years, applications of RNA aptamers to therapeutic drugs, diagnostic reagents, and test reagents have been drawing attention; some RNA aptamers have already been in clinical stage or actual use stage. In December 2004, the world's first RNA aptamer drug, Macugen, was approved as a therapeutic drug for age-related macular degeneration in the US. An RNA aptamer refers to an RNA that binds specifically to a target substance such as a protein, and can be prepared using the SELEX method (Systematic Evolution of Ligands by Exponential Enrichment) (Ellington et al., (1990) Nature, 346, 818-822; Tuerk et al., (1990) Science, 249, 505-510). The SELEX method is a method by which an RNA that binds specifically to a target substance is selected from a pool of about 1014 RNAs having different nucleotide sequences. The RNA used has a structure wherein a random sequence of about 40 residues is sandwiched by primer sequences. This RNA pool is allowed to associate with a target substance, and only the RNA that has bound to the target substance is recovered using a filter and the like. The RNA recovered is amplified by RT-PCR, and this is used as the template for the next round. By repeating this operation about 10 times, an RNA aptamer that binds specifically to the target substance can be acquired. If the RNA aptamer obtained promotes or inhibits a function of the target substance, this RNA aptamer will be applicable to pharmaceuticals and the like. Actually, RNA aptamers that bind specifically to the human translation initiation factor eIF4A (JP-A-2002-300885, Oguro et al., (2003) RNA 9, 394-407), eIF4E (JP-A-2004-344008, Mochizuki et al., (2005) RNA 11, 77-89), the bone metabolism-related receptor RANK (Receptor Activator of NF-κB, Mori et al., (2004) Nucleic Acids Res. 32, 6120-6128) and the like have been prepared using the SELEX method. An RNA aptamer that binds via an antigen recognition site of anti-DNA autoantibody has also been reported (Kim et al., (2003) Biochemical and Biophysical Research Communication 300, 516-523).